Characterization and evaluation are the functions where value is added to each accession. To avoid confusion when building capacity and in general use characterization means analyzing characters which are highly heritable, that can be seen easily by the eye (except molecular characters) and are expressed in all environments (color and morphology).
Evaluation referred to analyzing characters not as easily observed visually, such as pathogen or pest resistance or quality characters (oil or protein content, fiber content, fatty acid profile).
Curators must be aware of germplasm users’ (especially breeders) needs for information required to register new cultivars and focus on acquiring this information.
Agronomic and horticultural data are high priorities, but quality traits and disease-resistance data are also highly desired. Curators must also be aware of new diseases and respond quickly with evaluations for host-plant resistance. Some countries may not be devoting enough time/resources on evaluating germplasm for disease resistance or product quality, sometimes because a standard set of differential lines (isolates) for certain pathogens are lacking. The development of core (or sub-core) subsets is needed for more crop species.
Core subsets provide one rational mechanism for managing the genetic diversity within a collection.
Considering its global importance, it is striking that climate change is rarely mentioned in rationale or objectives for most gene banks. Curators and managers should assume a proactive role in preparing for this so as to minimize future crop production risk. Relevant germplasm should be acquired, new descriptor criteria (eg. resistance to drought, salinity, identification of stress tolerant genes) and techniques developed, and germplasm evaluated for those factors.
Examples of characterization & evaluation research to reduce risk
Analyses of phenotypic variation for 10,050 accessions of hexaploid cultivated oat were completed. Eight environmentally stable morphological characters differentiated the germplasm into 18 character states. Comparisons of oat diversity from different countries, among the major infraspecific groups and among Canadian oat cultivars registered between 1886 and 2002 were possible. The number of accessions in each morphological group was unevenly distributed with the 13 most frequent morphological groups representing 90% of the accessions. The most frequent morphological groups were identical to the most frequent types of Canadian oat cultivars. The greatest richness of diversity was found in oat from countries with temperate climates and intensive oat breeding programmes. The oat accessions comprised 8754 accessions of common hulled oat, 183 accessions of hull-less oat and 1168 accessions of red oat. For red oat (A. sativa (=A. byzantina C. Koch)), West Asia was richest in diversity. The USA could be considered a secondary centre of diversity for red oat and Canada a secondary centre of diversity for hull-less oat [93].
Modern molecular tools, such as AFLP (amplified fragment length polymorphism), SSR (simple sequence repeat) and DNA sequencing, are being used to characterize genetic diversity in plant and animal populations. Effective genetic analyses including Bayesian and stochastic modeling approaches are applied to partition genetic diversity within and among populations, whereas phylogenetic reconstruction methods retrace and illustrate molecular ancestry and relationships among the plant or animal groups of interest. Various diversity functions (Weitzman and Marker Estimated Kinship) help set priorities in plant and animal germplasm.
Germplasm characterization with molecular genetic markers has increased substantially. Although genetic marker information provides new insights into intrinsic genetic variability, the analyses and application of genetic markers involve many challenges.
National workshops to discuss these challenges and of integrating molecular data into practical gene bank research would be valuable. They could address the optimal types of marker, strengths/weaknesses for specific purposes, and the appropriate number of markers.
For some crops, it may be more efficient to involve industry or university partners or to farm-out genetic marker analyses rather than to develop the expertise “in-house.” This approach may increase interaction, take advantage of their expertise, increase utilization of collections and enhance capacity building within and among nations.
Diversity analyses based on molecular and agrobotanic studies have been conducted on wild and cultivated germplasm of flax [94,95,96] and oat [97,98,99] and several oilseed crucifer species [100]. These studies have resulted in more efficient germplasm conservation, and useful germplasm has been identified and integrated into breeding programs. Molecular analyses of the genetic diversity of wheat [101,102], soybean [103] and several native Canadian grass species [104,105,106,107,108,109]
have also justified the need for germplasm conservation.
AFLP analysis of 163 accessions of 25 Avena species with diverse geographic origins was completed. This analysis revealed 59.5% of the total AFLP variation resided among 25 oat species, 45.9% among six assessed sections of the genus, 36.1% among three existing ploidy levels, and 50.8% among eight defined genome types. All the species were clustered together according to their ploidy levels. The C genome diploids appeared to be the most distinct, followed by the Ac genome diploid A. canariensis. Analysis of AFLP similarity suggested that the AC genome tetraploid A. maroccana was likely derived from the Cp genome diploid A. eriantha and the As genome diploid A. wiestii, and might be the progenitor of the ACD genome hexaploids. These AFLP patterns are significant for our understanding of the evolutionary pathways of Avena species and genomes, for establishing reference sets of exotic oat germplasm, and for exploring new exotic sources of genes for oat improvement
[110]
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SSR analysis of 169 potato accessions representing diverse geographic origins was completed. This analysis showed 2.4% of the total SSR variation resided among accessions of different countries, 0.8% between Canadian and exotic potato accessions, 3% among Canadian accessions of four breeding periods defined, and 0.8% between Canadian heritage and non-heritage cultivars. These findings demonstrate the narrow genetic basis of potato germplasm and justify the need for broadening the improved potato gene pool. In spite of this, the most genetically distinct potato germplasm was identified for potato improvement [111].
The Microbial collection has contributed to research on several plant diseases and mycotoxins and provided information to national and international clients. Initial efforts to develop broad-spectrum diagnostic kits to detect Carlaviruses have been successful and prototype RT-PCR primer sets were used to detect and sequence two new highly divergent strains of Blueberry scorch virus in BC.
Evaluation of disease resistance in chickpea, canola and lentil relies on fungal isolates representing pathotypes, inoculation in growth chambers and verification with natural
infection in a cold-frame greenhouse. The genetics of resistance inheritance will requires the segregation of resistance in F1, F2 and backcross generations, populations of doubled haploid lines and/or recombinant inbred lines. Mapping of sclerotinia resistance involves microsatellite markers, and mapping of ascochyta resistance will use single nucleotide polymorphism.
Indoor disease screening methods were developed for lentil, canola, bean, sunflower, soybean and chickpea and a 4000 sq ft cold-frame greenhouse was built for screening under natural infection; QTL’s conferring sclerotinia resistance in canola were identified, and data on 1500 lentil accessions of which 35 were resistant to anthracnose were placed in GRIN-CA. Bioactive substances in diverse germplasm of pea and lentil have been isolated and identified.
Evaluation of canola (B. napus) germplasm for resistance to the fungal pathogen Sclerotinia sclerotiorum the cause of stem rot: QTLs conferring resistance to sclerotinia stem rot in the Chinese cultivar Zhong You 821 (ZY821) were mapped in 6 DH and 3 RIL populations. So far, QTLs have been identified on LG 1, 3 and 19. B. napus germplasm from PGRC, Dr. Rakow (AAFC, SRC), and the Nordic and Czech gene banks were screened for stem rot resistance; up to now, 10 new lines, obtained from Kashmir and Pakistan, have high levels of stem rot resistance. New methods for production of ascospores under controlled conditions and inoculation of canola stems, with ascospores on petals as nutrient source, were developed. Two sclerotinia nurseries were used to screen selected resistant and susceptible DH lines and germplasm under field conditions; however, inoculation of the soil with sclerotinia (survival structures) created variable inoculum pressure confounding the disease rating; consequently, inoculation of individual plants with ascospores on petals is now used. A micro array study of genes differentially up or down regulated in infected versus un-infected stem tissue, and susceptible versus resistant tissue were conducted and approximately 50 potential defence genes were identified.
Bioactive compound identification and seed oil evaluation uses various chromatographic techniques (medium pressure liquid, flash, thin-layer and gas) as well as electrospray – ionization, and matrix-assisted laser desorption/ionization mass spectrometry, and high-resolution NMR spectroscopy; concentrations will be assessed using HPLC with evaporative light scattering and photodiode array detectors.
Six chickpea seed samples supplied by Crop Development Centre, University of Saskatchewan were ground and flour samples (1-gram scale) extracted at ambient
temperature with 80% methanol. The crude extracts, treated briefly for 7 days with ordinary fluorescent light, were cleaned with Diaion HP-20 beads before soyasaponin analysis by HPLC. Defatted flours (100g) of the yellow field pea, green field pea, lentil, dry bean and soybean were processed in methanol with brief and 3 days of fluorescent light exposure in a chamber. The HP-20 methanol fractions from these extracts were isolated and examined by HPLC and electrospray LC/MS. The best source of dehydrosoyasaponin I was Amit chickpea followed by soybean and yellow field pea. Data was tabulated and submitted for inclusion into a PCT application.
Raw Amit chickpea flour (100 g scale), a controlled environment chamber maintained at 21C and equipped with 6x40 Watt fluorescent lights of the Cool White type (intensity of about 2250 lux at the centre of the chamber) proved to be an excellent visible light source for the generation of dehydrosoyasaponin I from soyasaponin VI. The utility of this chamber was demonstrated in two experiments with 80% methanol (48 hour light exposure) and two experiments with 60% ethanol (72 hour light exposure) as extraction solvent. Resulting Diaion HP-20 methanol (and ethanol) fractions enriched in dehydrosoyasaponin I (and soyasaponin I) were studied in detail by TLC, HPLC and electrospray LC/MS. Using these fractions (50 mg/injection), it was demonstrated that dehydrosoyasaponin I could be isolated in milligram quantities by medium pressure liquid chromatography with a custom-prepared reverse phase column of styrene/divinyl benzene (Source™ 15 RPC) and a gradient of ammonium hydroxide plus acetonitrile. Data was tabulated and submitted for inclusion into the PCT application.
Research on animals uses standard procedures for collection of germplasm from livestock and poultry breeds, extraction of proteins, Western blotting, immunolocalization, SDS-PAGE, 2-D electrophoresis, RT-PCR, and in situ hybridization. In vitro production of swine embryos depends on three techniques: in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos. Modifications to conditions and media are implemented to optimize embryo production. To transfer pig embryos, non-surgical methods are being developed. Modification of cryo-media and cryopreservation protocols are being developed to preserve bull, boar and rooster semen. Vitrification techniques are being used to preserve oocytes and embryos from different species.
Documentation
A computerized database management system (GRIN-CA) to handles the massive amount of data collected by PGRC, the Clonal Gene bank, the Nodes and the Animal Genetic Resources Program. The database software was acquired from the USDA, Database Management Unit, Beltsville, MD which means that all plant genetic resources in North America use the same database system. A new database structure is being developed for the animal program in cooperation with the Animal Genetic Resources Unit, Ft Collins, CO and EMBRAPA, Brazil. Combined, these systems with common programming language increase the programming capability and reduce the risk to loss data. The computer hardware/operating system used in Canada is a Sun/Solaris with an Oracle user interfaces supplemented by MVC web technology and traditional HTML. Timely back up of program language software and all data is essential to reduce risk of loss. Periodic placement of data at another location is highly desired.
Increased emphasis is being placed on digital imaging, especially when images of different organs (seed, flower, roots) are being captured so as to provide validity to descriptor characters. The next step will be to apply new technologies to numerically scan the images and generate numerical data as well. A major effort is still required to incorporate genetic marker data into GRIN-CA and this will be challenging.